Method for preventing or treating radiation and chemical damage

ABSTRACT

The present invention relates to use of plasminogen in prevention, treatment, amelioration and/or elimination of radiation and chemical damage and related diseases in a subject, and further provides a novel therapeutic strategy for treating different types of radiation and chemical damage.

FIELD OF THE INVENTION

The present invention relates to use of plasminogen or plasmin in prevention and/or treatment, amelioration and/or elimination of radiation and chemical damage and related diseases in a subject, and further provides a novel prevention and/or therapeutic strategy for preventing and/or treating different types of radiation and chemical damage.

BACKGROUND OF THE INVENTION

Radiation damage is damage to body tissue caused by radioactive rays exposure. Generally speaking, radioactive rays are high-energy electromagnetic waves or high-energy particles generated by natural or artificial energy sources. Instant exposure to high-dose radiation or long-term exposure to low-dose radiation may cause tissue damage. Chemical damage is local or systemic damage that occurs when chemicals contact human body. The degree of damage is related to the property of the chemicals, dosage, concentration, contact time and area, whether the treatment is timely and effective, and other factors. Clinically, radiotherapy or chemotherapy or a combination of the two is commonly used for treating or ameliorating tumor and cancer patients. Radiotherapy usually uses X-rays, gamma rays, neutrons and radiation from other sources to kill cancer cells or destroy genetic material within cells. Chemotherapy destroys cancer cell replication or proliferation by using cytotoxic drug alone or in combination. Almost all cancer patients exhibit severe side effects after going through one or more radiotherapy, chemotherapy or chemoradiotherapy, including mucosal ulcers, decreased immune function, bone marrow suppression, digestive disorders, inflammation, cardiotoxicity, nephrotoxicity, pulmonary fibrosis, phlebitis, neurotoxicity, hepatotoxicity, etc. . . . . Therefore, the prevention of the side effects of chemotherapy and radiotherapy and protection are crucial for cancer patients.

Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease which can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin and proteoglycans^([1]). In addition, plasmin can activate some pro-metalloproteinases (pro-MMP) to form active metalloproteinases (MMP). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis^([2, 3]). Plasmin is formed by proteolysis of plasminogen by two physiological PAs: tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Due to the relatively high level of plasminogen in plasma and other body fluids, it is conventionally believed that the regulation of the PA system is primarily achieved through PA synthesis and the level of activity. The synthesis of PA system components is strictly regulated by different factors such as hormones, growth factors and cytokines. In addition, there are also specific physiological inhibitors of plasmin and PA. The major inhibitor of plasmin is α2-antiplasmin. Some cells have on surfaces a direct hydrolytically active uPA specific cell surface receptor (uPAR)^([4, 5]).

Plasminogen (plg) is a single-stranded glycoprotein composed of 791 amino acids and has a molecular weight of approximately 92 kDa^([6,7]). Plasminogen is mainly synthesized in the liver and is abundantly present in the extracellular fluid. The amount of plasminogen in plasma is approximately 2 μM. Therefore, plasminogen is a huge potential source of proteolytic activity in tissues and body fluids^([8, 9]). Plasminogen has two molecular forms: glu-plasminogen and lys-plasminogen. Plasminogen of naturally secreted and uncleaved forms has an amino-terminal (N-terminal) glutamic acid, and thus it is referred to as glutamate-plasminogen. However, when plasmin is present, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys 76-Lys 77. Comparing with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg 560-Val 561 peptide bonds of these two forms of plasminogen can be cleaved by uPA or tPA, leading to the formation of disulfide-linked double-stranded protease plasmin^([10]). The amino-terminal portion of plasminogen contains five homotrimeric rings, i.e., the so-called kringles, and the carboxy-terminal portion contains a protease domain. Some kringles contain lysine bonding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. It has been recently discovered a fragment of plasminogen of 38 kDa, including kringle 1-4, which is an effective inhibitor of angiogenesis. This fragment is named angiosatin and can be produced by proteolysis of plasminogen by several protcascs.

The main substrate of plasmin is fibrin, and fibrinolysis is the key to prevent phathological thrombosis^([11]). Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycans, and gelatin, which indicates that plasmin also plays an important role in ECM remodeling^([7, 12, 13]). Indirectly, plasmin can also degrade other components of ECM by converting certain protease precursors into active proteases, including MMP-1, MMP-2, MMP-3 and MMP-9. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis^([14]). In addition, plasmin has the ability to activate certain potential forms of growth factors^([15-17]). In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.

We have surprisingly found in our research that plasminogen or plasmin has a significant therapeutic effect on radiation and chemical damage to the body and is of high safety. Therefore, the use of plasminogen or plasmin is a novel therapeutic strategy for the treatment of different types of radiation and chemical damage and related diseases.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to the use of plasminogen or plasmin in the preparation of a drug for treating and/or eliminating radiation damage and chemical damage and related diseases in a subject. The present invention also relates to the use of plasminogen or plasmin in the preparation of a drug for treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject. In one embodiment, the damage includes damage to a bone marrow hematopoietic system, skin, mucous membrane, an immune system and a reproductive system. In one embodiment, the damage includes damage to a liver, spleen, kidney, lung, gastrointestinal tract, thymus, bone marrow, testis and epididymis. In one embodiment, the damage is a decrease in general healthy conditions, systemic side effects and local side effects including acute side effects, long-term side effects and cumulative side effects caused by radiotherapy, chemotherapy or chemoradiotherapy. In one embodiment, the damage-related diseases include mucosal ulcers, decreased immune function, myelosuppression, digestive dysfunction, heart, liver, spleen, lung, kidney, ovarian, testicular toxicity dysfunctions, and neurotoxicity dysfunction. In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein including a plasminogen active fragment and still having plasminogen activity. In one embodiment, the plasminogen or plasmin is administered in combination with one or more other drugs or therapies, including anti-cancer drugs, anti-infective drugs, immunopotentiators, analgesics, nutrients and antidotes.

In one embodiment, the subject is a mammal, preferably a human.

In one embodiment, the subject lacks plasmin or plasminogen. Specifically, the lack is congenital, secondary and/or local.

In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein in which, based on SEQ ID NO: 2, 6, 8, 10 or 12, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid is added, deleted and/or substituted and which still has plasminogen activity. In one embodiment, the plasminogen is selected from a group consisting of glu-plasminogen, lys-plasminogen, mini-plasminogen, micro-plasminogen, and δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservatively substituted variant selected from variants of glu-plasminogen, lys-plasminogen, mini-plasminogen, δ-plasminogen and micro-plasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as plasminogen orthologs represented by SEQ ID NO: 2, for example, plasminogen orthologs from primates or rodents such as plasminogen orthologs from gorillas, rhesus monkeys, rats, cattle, horses and canines. Most preferably the amino acid sequence of the plasminogen of the present invention is as shown in SEQ ID NO: 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen or plasmin is administered by external, oral, systemic or topical administration. In one embodiment, the administration is via surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum. In one embodiment, the administration is external. In one embodiment, the topical administration is administering plasminogen or plasmin directly at the damaged area for prevention and/or treatment.

In one embodiment, the plasminogen is administered in combination with a suitable polypeptide or stabilizer. In one embodiment, the plasminogen is administered in a dosage of 0.0001 to 2000 mg/kg, 0.001 to 800 mg/kg, 0.01 to 600 mg/kg, 0.1 to 400 mg/kg, 1 to 200 mg/kg, 1 to 100 mg/kg, 10 to 100 mg/kg (calculated per kilogram of body weight) or 0.0001 to 2000 mg/cm², 0.001 to 800 mg/cm², 0.01 to 600 mg/cm², 0.1 to 400 mg/cm², 1 to 200 mg/cm², 1 to 100 mg/cm², 10 to 100 mg/cm² (calculated per square centimeter of surface area) per day, preferably it is repeated at least once, and preferably administered at least daily. In the case of topical administration, the above dosage may be further adjusted depending on the circumstances.

In another aspect, the present invention relates to plasminogen or plasmin for preventing and/or treating and/or eliminating radiation damage and chemical damage and related diseases in a subject, and a pharmaceutical composition containing plasminogen or plasmin for preventing and/or treating and/or eliminating radiation damage and chemical damage and related diseases in a subject. The present invention also relates to plasminogen or plasmin for preventing and/or treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject, and a pharmaceutical composition containing plasminogen or plasmin for preventing and/or treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject. In one embodiment, the damage includes damage to a bone marrow hematopoietic system, skin, mucous membrane, an immune system and a reproductive system. In one embodiment, the damage includes damage to a liver, spleen, kidney, lung, gastrointestinal tract, thymus, bone marrow, testis and epididymis. In one embodiment, the damage is a decrease in general healthy conditions, systemic side effects and local side effects including acute side effects, long-term side effects and cumulative side effects caused by radiotherapy, chemotherapy or chemoradiotherapy. In one embodiment, the damage-related diseases include mucosal ulcers, decreased immune function, myelosuppression, digestive dysfunction, heart, liver, spleen, lung, kidney, ovarian or testicular toxicity dysfunctions, and neurotoxicity dysfunction. In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein including a plasminogen active fragment and still having plasminogen activity. In one embodiment, the plasminogen or plasmin is administered in combination with one or more other drugs or therapies, including anti-cancer drugs, anti-infective drugs, immunopotentiators, analgesics, nutrients and antidotes.

In one embodiment, the subject is a mammal, preferably a human.

In one embodiment, the subject lacks plasmin or plasminogen. Specifically, the lack is congenital, secondary and/or local.

In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein in which, based on SEQ ID NO: 2, 6, 8, 10 or 12, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid is added, deleted and/or substituted and which still has plasminogen activity. In one embodiment, the plasminogen is selected from variants of glu-plasminogen, lys-plasminogen, mini-plasminogen, micro-plasminogen, and δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservatively substituted variant selected from conservatively substituted variants of glu-plasminogen, lys-plasminogen, mini-plasminogen, δ-plasminogen and micro-plasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as plasminogen orthologs represented by SEQ ID NO: 2, for example, plasminogen orthologs from primates or rodents such as plasminogen orthologs from gorillas, rhesus monkeys, rats, cows, horses and canines. Most preferably the amino acid sequence of the plasminogen of the present invention is as shown in SEQ ID NO: 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen or plasmin is administered by external, oral, systemic or topical administration. In one embodiment, the administration is via surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum. In one embodiment, the administration is external. In one embodiment, the topical administration is administering plasminogen or plasmin directly at the damaged area for the prevention and/or treatment.

In one embodiment, the plasminogen is administered in combination with a suitable polypeptide or stabilizer. In one embodiment, the plasminogen is administered in a dosage of 0.0001 to 2000 mg/kg, 0.001 to 800 mg/kg, 0.01 to 600 mg/kg, 0.1 to 400 mg/kg, 1 to 200 mg/kg, 1 to 100 mg/kg, 10 to 100 mg/kg (calculated per kilogram of body weight) or 0.0001 to 2000 mg/cm², 0.001 to 800 mg/cm², 0.01 to 600 mg/cm², 0.1 to 400 mg/cm², 1 to 200 mg/cm², 1 to 100 mg/cm², 10 to 100 mg/cm² (calculated per square centimeter of surface area) per day, preferably it is repeated at least once, and preferably administered at least daily. In the case of topical administration, the above dosage may be further adjusted depending on the circumstances.

In another aspect, the present invention relates to an article of manufacture or a kit containing plasminogen or plasmin for preventing and/or treating and/or eliminating radiation damage and chemical damage and related diseases in a subject, and an article of manufacture or a kit containing plasminogen or plasmin for preventing and/or treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject. In one embodiment, the damage includes damage to a bone marrow hematopoietic system, skin, mucous membrane, an immune system and a reproductive system. In one embodiment, the damage includes damage to a liver, spleen, kidney, lung, gastrointestinal tract, thymus, bone marrow, testis and epididymis. In one embodiment, the damage is a decrease in general healthy conditions, systemic side effects and local side effects including acute side effects, long-term side effects and cumulative side effects caused by radiotherapy, chemotherapy or chemoradiotherapy. In one embodiment, the damage-related diseases include mucosal ulcers, decreased immune function, myelosuppression, digestive dysfunction, heart, liver, spleen, lung, kidney, ovarian, testicular toxicity dysfunctions, and neurotoxicity dysfunction. In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein including a plasminogen active fragment and still having plasminogen activity. In one embodiment, the plasminogen or plasmin is administered in combination with one or more other drugs or therapies, including anti-cancer drugs, anti-infective drugs, immunopotentiators, analgesics, nutrients and antidotes.

In one embodiment, the article or kit further comprises a container containing one or more other drugs. The kit may also include an instruction further which states that the plasminogen or plasmin can be used for preventing and/or treating the damage and related diseases, and may further state that the plasminogen or plasmin is administered before, simultaneously with, and/or after the administration of other drugs or therapies.

In one embodiment, the subject is a mammal, preferably a human.

In one embodiment, the subject lacks plasmin or plasminogen. Specifically, the lack is congenital, secondary and/or local.

In one embodiment, the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity. In one embodiment, the plasminogen is a protein in which, based on SEQ ID NO: 2, 6, 8, 10 or 12, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid is added, deleted and/or substituted and which still has plasminogen activity. In one embodiment, the plasminogen is selected from a group consisting of glu-plasminogen, lys-plasminogen, mini-plasminogen, micro-plasminogen, and δ-plasminogen, or any combination thereof. In one embodiment, the plasminogen is a conservatively substituted variant selected from variants of glu-plasminogen, lys-plasminogen, mini-plasminogen, δ-plasminogen and micro-plasminogen. In one embodiment, the plasminogen is a natural human plasminogen, such as plasminogen orthologs represented by SEQ ID NO: 2, for example, plasminogen orthologs from primates or rodents such as plasminogen orthologs from gorillas, rhesus monkeys, rats, cows, horses and canines. Most preferably the amino acid sequence of the plasminogen of the present invention is as shown in SEQ ID NO: 2, 6, 8, 10 or 12.

In one embodiment, the plasminogen or plasmin is administered by external, oral, systemic or topical administration. In one embodiment, the administration is via surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or through the rectum. In one embodiment, the administration is external. In one embodiment, the topical administration is administering plasminogen or plasmin directly at the damaged area for the prevention and/or treatment.

In one embodiment, the plasminogen is administered in combination with a suitable polypeptide or stabilizer. In one embodiment, the plasminogen is administered in a dosage of 0.0001 to 2000 mg/kg, 0.001 to 800 mg/kg, 0.01 to 600 mg/kg, 0.1 to 400 mg/kg, 1 to 200 mg/kg, 1 to 100 mg/kg, 10 to 100 mg/kg (calculated per kilogram of body weight) or 0.0001 to 2000 mg/cm², 0.001 to 800 mg/cm², 0.01 to 600 mg/cm², 0.1 to 400 mg/cm², 1 to 200 mg/cm², 1 to 100 mg/cm², 10 to 100 mg/cm² (calculated per square centimeter of surface area) per day, preferably it is repeated at least once, and preferably administered at least daily. In the case of topical administration, the above dosage may be further adjusted depending on the circumstances.

The present invention explicitly covers all combinations of technical features pertaining to embodiments of the present invention, and these combined technical solutions have been explicitly disclosed in the present application, just as that the above technical solutions have been independently and explicitly disclosed. In addition, the present invention also explicitly covers all sub-combinations of all embodiments and elements thereof, and is disclosed herein, just as that each of such sub-combinations is independently and explicitly disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

“Radiation damage”, also referred to as “radiation injury”, is damage (injury) to whole body or local organs or tissues caused by radioactive rays exposure. Generally speaking, radioactive rays are high-energy electromagnetic waves or high-energy particles generated by natural or artificial energy sources. Instant exposure to high-dose radiation or long-term exposure to low-dose radiation may cause organ or tissue damage. “Chemical damage” is local or systemic damage (injury) that occurs when chemicals contact human body. The degree of damage is related to the property of the chemicals, dosage, concentration, contact time and area, whether the treatment is timely and effective, and other factors.

Radiation damage and chemical damage can be manifested as damage to organs, tissues and functions of the body, for example, damage to physiological structure and function of the bone marrow hematopoietic system, damage to physiological structure and function of the skin and mucous membranes, damage to physiological structure and function of the immune system, and damage to physiological structure and function of the reproductive system. In one embodiment, the damage includes damage to the physiological structure and function of a liver, spleen, kidney, lung, gastrointestinal tract, thymus, bone marrow, testis and epididymis. The damage-related diseases are manifested as damaged organ and tissue dysfunction, such as mucosal ulcers, decreased immune function, myelosuppression, digestive dysfunction, heart, liver, spleen, lung, kidney, ovarian, testicular toxicity dysfunctions, and neurotoxicity dysfunction.

“Radiation therapy” and “radiotherapy” can be used interchangeably. The treatment with chemical drugs is generally referred to as “chemotherapy” for short. “Chemoradiotherapy” refers to a combination of radiotherapy and chemotherapy. Clinically, radiotherapy or chemotherapy or a combination of the two is commonly used for treating or ameliorating tumor and cancer patients. Radiotherapy usually uses X-rays, gamma rays, neutrons and radiation from other sources to kill cancer cells or destroy genetic material within cells. Chemotherapy destroys cancer cell replication or proliferation by using cytotoxic drug alone or in combination. Normal cells are also damaged during radiotherapy and cannot repair themselves. Side effects may occur during radiotherapy, including skin irritation, hair loss in the treatment area, and bone marrow damage.

“Chemotherapy” refers to the use of cytotoxic drug alone or in combination to kill cancer cells. Similar to radiotherapy, cancer cells can be damaged and eventually die; however, healthy cells are affected during the self-repair process after chemotherapy. Cytotoxic drugs act by interfering with the differentiation and proliferation capabilities of growing cells. Therefore, in addition to cancer cells, other cells that normally differentiate and grow rapidly are also affected. For example, they may affect the hematopoietic function of the bone marrow, causing bone marrow suppression. In addition, they may affect the digestive tract, the inner walls of the oral cavity and the reproductive system cells, and affect the hair follicles, causing diarrhea, oral pain and hair loss.

“The side effects of chemoradiotherapy” refer to side effects associated with chemoradiotherapy, usually occurring at different stages, such as during the treatment (acute side effects), months or years after the treatment (long-term side effects), or after repeated treatment (accumulated side effects). The nature, severity and length of side effect depend on the organ being treated, the treatment itself (radiotype, dose, grade, combined chemotherapy) and the patient.

Most of the side effects are predictable and expectable. The side effects of radiotherapy are usually limited to a topical area of the patient where treatment is received. A goal of the modern radiotherapy method is to minimize side effects and help patients understand and manage those unavoidable side effects.

Myelosuppression is one of the many side effects of radiotherapy and chemotherapy. The result is reduced blood cell production, including red blood cells, white blood cells, and platelets. As a result, the patient is fatigued due to anaemia and becomes susceptible to infection due to leucopenia, and due to thrombocytopenia, bruises and bleeding are more likely to occur when there is a wound.

“General health condition” is a healthy standard, and it refers to that an individual can eat, talk and participate in social activities which help to satisfy basic recreation based on that the body, related tissues and organs have no active disease, discomfort and anxiety. Main signs of general health include physical health and mental health.

“Plasmin” is a very important enzyme that exists in the blood and can hydrolyze fibrin clots into fibrin degradation products and D-dimers.

“Plasminogen” is a zymogen form of plasmin. It consists of 810 amino acids calculated from the natural human plasminogen amino acid sequence (SEQ ID NO: 4) containing a signal peptide according to the sequence in Swiss prot and has a molecular weight of about 92 kD. It is a glycoprotein synthesized mainly in the liver and capable of circulating in the blood, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 3. A full-length plasminogen includes seven domains: a serine protease domain at C-terminal, a Pan Apple (Pap) domain at N-terminal, and five Kringle domains (Kringle 1 to 5). Referring to the sequence in Swiss prot, the signal peptide includes residues Met 1-Gly 19, Pap includes residues Glu 20-Val 98, Kringle 1 includes residues Cys 103-Cys 181, Kringle 2 includes residues Glu 184-Cys 262, Kringle 3 includes residues Cys 275-Cys 352, Kringle 4 includes residues Cys 377-Cys 454, and Kringle 5 includes residues Cys 481-Cys 560. According to NCBI data, the serine protease domain includes residues Val 581-Arg 804.

Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing signal peptide of 19 amino acids), and the cDNA sequence encoding this sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In vivo, lys-plasminogen, which is formed by hydrolysis at the 76th to 77th amino acids of the glu-plasminogen, is also present and is shown in SEQ ID NO: 6, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 5. δ-plasminogen is a fragment of a full-length plasminogen that lacks the Kringle 2-Kringle 5 structure and contains only the Kringle 1 and serine protease domains^([18, 19]) The amino acid sequence (SEQ ID NO: 8) of δ-plasminogen has been reported^([19]), and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 7. Mini-plasminogen consists of Kringle 5 and serine protease domains. It has been reported that it includes residues Val 443-Asn 791 (taking the glu-residue of the glu-plasminogen sequence not containing signal peptide as the starting amino acid)^([20]). The amino acid sequence is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9. Micro-plasminogen only includes the serine protease domain, and it has been reported that the amino acid sequence includes residues Ala 543-Asn 791 (taking the glu-residue of the glu-plasminogen sequence not containing signal peptide as the starting amino acid)^([21]), It has also been reported in the patent document CN 102154253A that the sequence includes residues Lys 531-Asn 791 (taking the glu-residue of the glu-plasminogen sequence not containing signal peptide as the starting amino acid), and the sequence of the present patent application refers to the Patent Literature CN 102154253A. The amino acid sequence is shown in SEQ ID NO: 12, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 11.

In the present invention, “plasmin” is used interchangeably with “fibrinolysin” and “fibrinoclase”, and the terms have the same meaning; and “plasminogen” is used interchangeably with “fibrinolytic zymogen” and “fibrinoclase zymogen”, and the terms have the same meaning.

In the course of circulation, plasminogen adopts a closed inactive conformation. However, when it is bound to a thrombus or cell surface, it becomes an active plasmin in an open conformation under the mediation of a plasminogen activator (PA). The active plasmin can further hydrolyze the fibrin clot to fibrin degradation products and D-dimers, and further dissolves the thrombus. The Pap domain of plasminogen contains an important determinant that maintains plasminogen in an inactive closed conformation, whereas the KR domain is capable of binding to lysine residues present on receptors and substrates. It is known that a variety of enzymes can act as plasminogen activators, including tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), kallikrein and coagulation factor XII (Hageman factor).

“Plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein. The technical solution of the present invention relating to plasminogen covers a technical solution replacing plasminogen with a plasminogen active fragment. The plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen. Preferably, the plasminogen active fragment of the present invention comprises SEQ ID NO: 14, or an amino acid sequence having an amino acid sequence identity of at least 80%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 14. Therefore, plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still having plasminogen activity.

At present, methods for testing plasminogen and its activity in blood include detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t-PAAg), detection of plasma tissue plasminogen activity (plasminogen A), detection of plasma tissue plasminogen antigen (plasminogen Ag), detection of plasma tissue plasminogen activator inhibitor activity, detection of plasma tissue plasminogen activator inhibitor antigen, and detection of plasma plasmin-antiplasmin composite (PAP). The most commonly used detection method is the chromogenic substrate method. That is, streptokinase (SK) and chromogenic substrate are added to the subject plasma, the plasminogen in the subject plasma is converted to PLM under the action of SK and PLM acts on chromogenic substrate. Then a spectrophotometer is used for measurement, and the increase in absorbance is proportional to the plasminogen activity. In addition, plasminogen activity in blood can also be measured by an immunochemical method, a gel electrophoresis, immunoturbidimetry, a radioimmuno-diffusion method, etc. . . . .

“Orthologues or orthologs” refer to homologs between different species, including both protein homologs and DNA homologs, and are also known as orthologous homologs and vertical homologs. The term specifically refers to proteins or genes that have evolved from the same ancestral gene in different species. The plasminogen of the present invention includes human natural plasminogen, and also includes orthologues or orthologs of plasminogen derived from different species and having plasminogen activity.

A “conservative substitutional variant” refers to a change in a given amino acid residue without changing the overall conformation and function of a protein or enzyme, which includes and is not limited to substituting amino acids in the amino acid sequence of the parental protein with amino acids of similar properties (for example, acidity, basicity, hydrophobicity, etc.). Amino acids having similar properties are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and are interchangeable. Similarly, isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different, for example, 70% to 99% similarity (identity) based on the MEGALIGH algorithm. A “conservative substitution variant” also includes a polypeptide or enzyme having 60% or more amino acid identity determined by BLAST or FASTA algorithm, preferably 75% or more, more preferably 85% or more, most preferably 90% or more, and having the same or substantially similar properties or functions as compared with natural or parental proteins or enzymes.

“Isolated” plasminogen or plasmin refers to plasminogen or plasmin protein that is isolated and/or recovered from its natural environment. In some embodiments, the plasminogen or plasmin will be purified (1) to a purity more than 90%, more than 95%, or more than 98% (by weight), for example, more than 99% (by weight), as determined by Lowry method, (2) to an extent enough to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (3) to homogeneity, the homogeneity being determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue or silver staining under reducing or non-reducing conditions. Isolated plasminogen or plasmin also includes plasminogen or plasmin prepared from recombinant cells by bioengineering techniques and separated by at least one purification step.

The terms “polypeptide”, “peptide” and “protein” are interchangeable herein and refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to, fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences (with or without N-terminal methionine residues), etc. . . . .

The “percentage (%) of amino acid sequence identity” with reference to polypeptide sequence is defined as a percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues with reference to polypeptide sequence after a gap is introduced as necessary to achieve maximum percentage of sequence identity and when no conservative substitution is considered as part of sequence identity. Comparisons for the purpose of determining percentage of amino acid sequence identity may be achieved in a variety of ways within the technical scope in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum contrast over the full length of the sequences being compared. However, for purposes of the present invention, the percentage of amino acid sequence identity is generated using the sequence comparison computer program ALIGN-2.

In the case of comparing amino acid sequences using ALIGN-2, the percentage of amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as a given amino acid sequence A having or containing a percentage of amino acid sequence identity relative to, to, or with respect to a given amino acid sequence B) is calculated as follows:

Fraction X/Y×100

wherein X is a specific number of identically matched amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignment of the program, and Y is a total number of amino acid residues in B. It should be understood that, when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity of A relative to B will not be equal to the percentage of amino acid sequence identity of B relative to A. All percentages of amino acid sequence identity used herein are obtained using ALIGN-2 computer program as described in the previous paragraph, unless specifically stated otherwise.

As used herein, the terms “treating”, “treatment” and “eliminating” refer to obtaining a desired pharmacological and/or physiological effect. The effect may be complete or partial prevention of a disease or its symptoms, and/or partial or complete cure of a disease and/or its symptoms, and includes: (a) preventing the occurrence of a disease in a subject which may have the cause of the disease but not yet diagnosed as having the disease; (b) inhibiting the disease, that is, holding back its formation; and (c) alleviating the disease and/or its symptoms, that is, causing the disease and/or its symptoms to subside.

The terms “individual”, “subject” and “patient” are interchangeable herein and refer to mammals, including but not limited to rats (rats, mice), non-human primates, humans, canines, cats, hoofed animals (such as horses, cattle, sheep, pigs, goats) and the like.

A “therapeutically effective amount” or an “effective amount” refers to an amount of plasminogen sufficient to achieve the prevention and/or treatment of a disease when it is administered to a mammal or other subjects to treat a disease. The “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated and the age, weight, etc. . . . .

Preparation of Plasminogen or Plasmin of the Present Invention

Plasminogen or plasmin can be isolated and purified from nature for further therapeutic uses, and can also be synthesized by standard chemical peptide synthesis techniques. In chemical synthesis of polypeptides, polypeptides can be synthesized via a liquid or a solid phase. Solid phase polypeptide synthesis (SPPS) (in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a method suitable for the chemical synthesis of plasminogen or plasmin. Various forms of SPPS such as Fmoc and Boc can be used to synthesize plasminogen or plasmin. Techniques for solid phase synthesis are described in Barany and Solid-Phase Peptide Synthesis, pages 3 to 284; The Peptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2^(nd) ed. Pierce Chem. Co., Rockford, III. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero J A et al., 2005 Protein Pept Lett. 12: 723-8. In short, small insoluble porous beads are treated with a functional unit on which a peptide chain is built. After repeated cycles of coupling/deprotection, the attached solid-phase free N-terminal amine is coupled to a single N-protected amino acid unit. Then the unit is deprotected to expose new N-terminal amines that can be attached to other amino acids. The peptide remains fixed on the solid phase before it is cut off.

Standard recombinant methods can be used to produce the plasminogen of the present invention. For example, a nucleic acid encoding a plasminogen is inserted into an expression vector such that it is operably linked to a regulatory sequence in the expression vector. Expression regulatory sequences include, but are not limited to, promoters (for example, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression regulation can be a eukaryotic promoter system in a vector which is capable of transforming or transfecting eukaryotic host cells (for example, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for a high-level expression of the nucleotide sequence and collection and purification of plasminogen.

Suitable expression vectors are usually replicated in the host organism as episomes or as an integral part of the host chromosomal DNA. In general, an expression vector contains a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to help to test cells that have been transformed with the desired DNA sequence of foreign sources.

Escherichia coli is an example of a prokaryotic host cell that can be used to clone the subject antibody-encoding polynucleotide. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be generated in these prokaryotic hosts and will usually contain expression regulatory sequences (e.g., origins of replication) that are compatible with the host cells. In addition, there will be many well-known promoters, such as lactose promoter system, tryptophan (trp) promoter system, β-lactamase promoter system, or promoter system from phage lambda. The promoters usually regulate expressions to initiate and complete transcription and translation optionally in a case of manipulation of gene sequences and having ribosome binding site sequences and the like.

Other microorganisms, such as yeasts, can also be used for expression. Yeast (such as S. cerevisiae) and Pichia are examples of suitable yeast host cells, where suitable vectors have expression regulatory sequences (such as promoters), origins of replication, termination sequences and the like as needed. Typical promoters include 3-phosphoglycerate kinase and other saccharolytic enzymes. Inducible yeast is initiated by promoters that specifically include enzymes derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

In addition to microorganisms, mammalian cells (eg, mammalian cells cultured in in vitro cell culture) can also be used to express and produce the protein of the invention (eg, polynucleotides encoding the subject protein). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors used for these cells may contain expression regulatory sequences such as origins of replication, promoters and enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Examples of suitable expression regulatory sequences include promoters derived from white immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148: 1149 (1992).

Once synthesized (chemical or recombinant), the plasminogen or plasmin of the present invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis and the like. The plasminogen or plasmin is substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or purer, for example, free of contaminants such as cell debris; macromolecules other than the plasminogen, etc. . . . .

Pharmaceutical Formulations

Therapeutic formulations may be prepared by mixing plasminogen or plasmin having required purity with optional medicinal carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16^(th) ed., Osol, A. ed. (1980)) to form lyophilized formulations or aqueous solutions. Acceptable carriers, excipients and stabilizers are non-toxic to recipients at the employed dosages and concentrations, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexanediamin chloride; benzalkonium chloride, benzoxonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; saccharides such as sucrose, mannitol, fucose, or sorbitol; salt foring counter ions such as sodium; metal complexes (e.g., zinc-protein complex); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Formulations of the present invention may also contain one or more active compounds required for specific conditions to be treated, preferably those that are complementary in activity and have no side effects with each other, for example, anti-tumor drugs, anti-cancer drugs, anti-infective drugs, immune enhancers, pain killers, nutrients, antidotes, etc. . . . .

Plasminogen or plasmin of the present invention may be encapsulated in microcapsules prepared by, such as, coacervation technology or interfacial polymerization. For example, it can be placed in a colloidal pharmaceutical delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16^(th) edition, Osol, A. Ed. (1980).

Plasminogen or plasmin of the present invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after freeze drying and re-preparation.

Plasminogen or plasmin of the present invention can be used to prepare sustained-release formulations. Suitable examples of sustained-release formulations include solid hydrophobic polymer semi-permeable matrices having certain shapes and containing glycoproteins, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., ibid), or degradable lactic acid-glycolic acid copolymers such as Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for 100 days or more, while some hydrogels release proteins for a shorter time. A rational strategy for protein stabilization can be designed based on relevant mechanisms. For example, when the mechanism of agglomeration is found to be the formation of intermolecular S—S bonds through thiodisulfide interchange, stability can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling humidity, using suitable additives, and developing specific polymer matrix compositions.

Administration and Dosage

The administration of the pharmaceutical composition of the present invention can be realized in different ways, for example, by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via carotid), intramuscular, intranasal, topical or intradermal administration or spinal cord or brain delivery. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of active agents and preservatives and other isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include liquid and nutrient supplements, electrolyte supplements, etc. . . . . Preservatives and other additives may also be present, for example, antimicrobials, antioxidants, chelating agents, inert gases, etc. . . . .

Medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical field, the dose for any patient depends on a variety of factors, including the patient's body size, body surface area, age, the specific compound to be administered, gender, frequency and route of administration, overall health, and other medications administered simultaneously. The dose of the pharmaceutical composition containing plasminogen of the present invention may be, for example, about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject's body weight per day. For example, the dosage may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1 to 50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also included in the scope of the present invention, especially considering the aforementioned factors. Intermediate dosages in the aforementioned ranges are also included in the scope of the present invention. A subject is administered such doses daily, every other day, weekly or any other schedule determined by empirical analysis. Exemplary dosage schedules include 1 to 10 mg/kg for consecutive days. In the drug administration process of the present invention, real-time evaluation and periodic evaluation of the therapeutic effects and safety of radioactive and chemical injuries and related diseases are required.

Therapeutic Efficacy and Treatment Safety

One embodiment of the present invention relates to the judgment of therapeutic efficacy and treatment safety after treating a subject with plasminogen or plasmin. Methods for judging the therapeutic efficacy include, but are not limited to: 1) examination of recovery of the immune system, specifically, for example, recovery of leukocyte and platelet count, and the subject being expected to recover to normal range or improve after receiving the plasminogen or plasmin treatment of the present invention, such as leukocytes recovering to 4 to 10×10⁹/L and platelets recovering to 100 to 300×10⁹/L; 2) improvement of the poor performance of the digestive system, including improved anorexia, nausea, vomiting, diarrhea, constipation and other symptoms; 3) improvement of functions of various organs of the organism, including improvement of liver function such as alanine aminotransferase (ALT), total bilirubin level, renal function, etc., specifically, the subject being expected to return to normal range or improve after receiving the plasminogen or plasmin treatment of the present invention, for example, alanine aminotransferase (ALT): 0 to 40 μ/L, total bilirubin: 3.4 to 20.5 μmol/L; 4) amelioration of phlebitis, ulcers and other symptoms. In addition, the present invention also relates to the judgment of the safety of the therapeutic regimen in the process of treatment and after treatment of a subject using plasminogen or plasmin, including but not limited to counting the subject's serum half-life, half-life of treatment, medium toxic dose (TD50), median lethal dose (LD50), or observing various adverse events such as sensitization reactions occurred during or after treatment.

Articles of Manufacture or Kits

One embodiment of the present invention relates to an article of manufacture or a kit containing plasminogen or plasmin of the present invention for treating radiation damage and chemical damage and related diseases. The article preferably includes a container, label or package insert. Suitable containers include bottles, vials, syringes, etc. . . . . The container may be made of various materials such as glass or plastic. The container contains a composition that is effective for treating a disease or disorder of the invention and has a sterile access (for example, the container may be an intravenous solution bag or vial containing a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is plasminogen or plasmin. The container or the attached label indicates that the composition is used to treat the radioactive and chemical damage and related diseases of the present invention. The article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and dextrose solution. It may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes. In addition, the article contains a package insert with instructions for use, including, for example, instructing a user of the composition to administer a plasminogen or plasmin composition and other medications for treating accompanying diseases to a patient.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a change in body weight of mice irradiated with 5.0 Gy X-ray after administration of plasminogen.

FIG. 2 shows observed result of HE staining of kidneys 10 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 3 shows observed result of immunohistochemical staining of macrophage marker F4/80 of kidneys 10 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 4 shows observed result of HE staining of duodenum 10 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 5 shows observed result of immunohistochemical staining of macrophage marker F4/80 of duodenum 10 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 6 shows observed result of immunohistochemical staining of macrophage marker F4/80 of livers 10 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 7 shows observed results of HE staining of livers 1, 4, 7 and 14 days after administration of plasminogen in mice irradiated with 5.0 Gy X-ray.

FIG. 8 shows a change in body weight of 10 mg/kg cisplatin-chemotherapy-injured model mice 7 days after administration of plasminogen.

FIG. 9 shows observed result of HE staining of kidneys 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

FIG. 10 shows observed result of immunohistochemical staining of fibrin in kidneys 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

FIG. 11 shows observed result of Bcl-2 immunohistochemical staining of kidneys 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

FIG. 12 shows observed result of HE staining of livers 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

FIG. 13 shows observed result of immunohistochemical staining of fibrin in livers 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

FIG. 14 shows observed result of HE staining of testes and epididymides 7 days after administration of plasminogen in 10 mg/kg cisplatin-chemotherapy-injured model mice.

EXAMPLES

Materials and Methods:

Radiation Damage Model:

Experimental Animals:

SPF level healthy male C57 mice of 7 to 8 weeks old were used to study histopathological changes of organs such as spleen, liver and kidney after 5.0 Gy irradiation. The animals were randomly divided into three groups, blank control group, simple irradiation group, and plasminogen administration group after 7 days of adaptive feeding.

Experimental Method:

A linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, the absorbed dose was 5.0 Gy which is sub-lethal dose irradiation, and this was used to study histopathological changes of organs such as spleen, liver and kidney, hematopoietic and immunological functions and free radical detection. The source skin distance was 100 cm, the irradiation area was 30 cm×30 cm, and the normal control group was covered with lead for protection. After irradiation, the mice were administered till death or were killed at a specific time, and the control group was given a corresponding volume of solvent. The general condition of the exposed mice was observed, and the histopathological changes of HE staining of liver, intestine and kidney tissues and immunohistochemical staining of F4/80 were observed under light microscope.

Observation Indexes:

1. Histopathological observation of liver, intestine and kidney:

After eyes and blood were taken and the mice were killed, the spleen liver, intestine and kidney tissues were taken and fixed in 10% neutral formalin, dehydrated by gradient alcohol, transparentized in xylene, paraffin-embedded, and paraffin sectioned (sheet thickness of 5 μm). After normal HE staining, they were sealed by neutral gum and the histopathological changes were observed under microscope.

2. F4/80 immunohistochemical staining observation of liver, intestine and kidney.

Liver, intestine, and kidney were paraffin sectioned (sheet thickness of 5 μm) and immunohistochemically stained, and then expression in tissues were observed.

Chemical Damage Model:

Experimental Animals:

SPF level healthy male C57 mice of 7 to 8 weeks old were used to observe side effects of anti-cisplatin. The animals were randomly divided into three groups, blank control group, simple model group, and plasminogen administration group after 7 days of adaptive feeding.

Experimental Method:

Cisplatin group: Normal saline for cisplatin ampoule was formulated into an aqueous solution having a mass concentration of 1 mg/ml and was administered by intraperitoneal injection at 3.5 ml/kg body weight. Mice of the control group received intraperitoneal injection of an equal volume of normal saline each time. Model was made after 5 days of continuous administration, and after making the model, the treatment group was given plasminogen at 1 mg/body while the control group and the model group were given the same volume of solvent, and the animals were killed on the 7th day after the administration of plasminogen. Blood and kidney tissues were taken for index test.

Observation Indexes:

1. Weigh change curve: The body weight of the mice was measured daily before administration and the change in body weight in each group of mice was observed.

2. Kidney coefficient: After the mice were killed, the kidney tissues were immediately taken and freshly weighed to calculate the kidney coefficient [kidney coefficient Z (organ mass/body weight)×100%].

3. Kidney tissue HE observation: Kidney tissues were fixed in 10% neutral formalin, dehydrated by gradient alcohol, transparentized in xylene, paraffin-embedded, and paraffin sectioned (sheet thickness of 5 μm). After normal HE staining, they were sealed by neutral gum and the histopathological changes were observed under microscope.

4. Animal thymus coefficient and spleen coefficient: After the mice were killed, the thymuses and spleens were immediately taken and freshly weighed to calculate the thymus coefficient and spleen coefficient [organ coefficient Z (organ mass/body weight)×100%].

5. Testis and epididymis weight: After mice of each group were killed, testes and epididymides were taken and weighed for statistical analysis.

6. Testicular tissue morphology observation: After the testicular tissues were conventionally fixed, dehydrated, embedded and sectioned, HE staining was used to observe the morphological changes of the testicular tissues.

Example 1: Effect of Plasminogen on Mice Body Weight after 5.0 Gy X-Ray Irradiation

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The body weights of the mice were measured and recorded on the 0^(th), 5^(th), 12^(th), and 21^(st) days of the experiment.

There was no obvious difference in body weight data between the solvent PBS control group and the plasminogen administration group on the 0^(th), 5^(th), 12^(th), and 21^(st) days (FIG. 1). This shows that X-ray radiation and administration do not affect the body weights of the mice.

Example 2: Protective Effect of Plasminogen on Kidneys of Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The mice were killed and dissected on the 21^(st) day and the kidneys were fixed in 10% neutral formalin for 24 to 48 hours. The fixed kidney tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The result showed that glomerular atrophy (*) and kidney tubular protein cast (□) were observed in the solvent PBS control group (FIG. 2A), while for the plasminogen administration group (FIG. 2B), glomerular capillary lumen was unobstructed and balloon lumen was clearly visible. The damage to the kidneys of the plasminogen administration group was notably less than that of the solvent PBS control group, which indicates that the injection of plasminogen can promote the repair of renal damage caused by X-ray radiation.

Example 3: Plasminogen Promoting Repair of Renal Inflammation in Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The mice were killed and dissected on the 21^(st) day and the kidneys were fixed in 10% neutral formalin for 24 to 48 hours. The fixed kidney tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to Tris-EDTA repair for 30 minutes, cooled at room temperature for 20 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse F4/80 antibody (Abcam) and were washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abcam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

The F4/80 macrophage marker can indicate the degree and stage of an inflammatory reaction. The result showed that the expression level of mouse macrophage marker F4/80 in the solvent PBS control group (FIG. 3A) was higher than that in the plasminogen administration group (FIG. 3B), indicating that inflammation of the kidney tissues of animals was significantly reduced after plasminogen was given. Quantitative analysis results were consistent with microscopic observations and statistical differences were significant (FIG. 3C), which indicates that plasminogen can promote repair of renal inflammation caused by X-ray radiation.

Example 4: Protective Effect of Plasminogen on Duodenum of Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0th day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The mice were killed and dissected on the 21^(st) day and the duodenums were fixed in 10% neutral formalin for 24 to 48 hours. The fixed duodenum tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The result showed that for the solvent PBS control group (FIG. 4A), local intestinal mucosal epithelium was detached and degenerated to necrosis, and normal mucosal structure at the detached place disappeared, while for the plasminogen administration group (FIG. 4B), red-stained refractive striated border was visible, goblet cells were visible on the mucosal epithelium, the villi center was lamina propria with a clear structure and distinct layers. The damage to the duodenums of the plasminogen administration group was notably less than that of the solvent PBS control group, which indicates that the injection of plasminogen can promote the repair of duodenal damage caused by X-ray radiation.

Example 5: Plasminogen Promoting Repair of Duodenal Inflammation in Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The mice were killed and dissected on the 21^(st) day and the duodenums were fixed in 10% neutral formalin for 24 to 48 hours. The fixed duodenum tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to Tris-EDTA repair for 30 minutes, cooled at room temperature for 20 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse F4/80 antibody (Abeam) and were washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abeam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

The result showed that the expression level of mice F4/80 in the solvent PBS control group (FIG. 5A) was higher than that in the plasminogen administration group (FIG. 5B), which indicates that inflammation of the duodenums was significantly reduced after plasminogen was given, showing that plasminogen can promote repair of duodenal inflammation caused by X-ray radiation.

Example 6: Plasminogen Promoting Repair of Liver Inflammation in Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 10 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 10 days. After the completion of the administration, the animals were observed for 11 days, and the entire experiment period was 21 days. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The mice were killed and dissected on the 21^(st) day and the livers were fixed in 10% neutral formalin for 24 to 48 hours. The fixed liver tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to Tris-EDTA repair for 30 minutes, cooled at room temperature for 20 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse F4/80 antibody (Abcam) and were washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abcam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

The F4/80 immunohistochemical result showed that the expression level of mouse macrophage marker F4/80 in the solvent PBS control group (FIG. 6A) was higher than that in the plasminogen administration group (FIG. 6B) after the irradiation with 5.0 Gy X-ray and the establishment of the model, which indicates that inflammation of the liver tissues of the animals was significantly reduced after plasminogen was given.

Example 7: Protective Effect of Plasminogen on Livers of Mice Irradiated with 5.0 Gy X-Rays

In the present experiment, 24 healthy male C57 mice aged 6 to 8 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 12 mice for each group. After grouping, a radiation damage model was established and a linear accelerator 6 MV X-ray 5.0 Gy was used to irradiate mice uniformly of the whole body for a single time with an absorbed dose rate of 2.0 Gy/min, and the absorbed dose was 5.0 Gy (irradiation for 2.5 minutes). After the model was established, plasminogen was given within 3 hours. The plasminogen administration group was administered via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Radiation was given and plasminogen or solvent PBS was given from the first day, and the administration period was 14 days. 3 animals were killed respectively on the first day, the 4^(th) day, the 7^(th) day and the 14^(th) day after irradiation. The livers of the mice were fixed in 10% neutral formalin for 24 to 48 hours. The fixed liver tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The result showed that for livers of the solvent PBS control group (FIG. 7a-d ) on the first day, the hepatic cords spread radially around the central vein with regular texture and clear hepatic sinusoid, some hepatic cells were degenerated to necrosis, karyolysis was present and cytoplasm was lightly stained (←). On the 4^(th) day, mild inflammatory cell infiltration occurred in liver sinusoids (▾), and hepatic sinusoids narrowed. On the 7^(th) day, hepatocyte necrosis was further aggravated, and liver cells showed mild watery degeneration (□), cytoplasm was dissolved and hepatic cord disorders occurred. On the 14^(th) day, livers had improved greatly. The hepatic cords were regular and the hepatic sinusoids were clear. However, there was still a small amount of inflammatory cells infiltrating into the hepatic sinusoids near the central veins.

For the plasminogen administration group (FIG. 7e-g ), on the first day, the hepatic cords were regular, the hepatic sinusoids were clear and there is a small amount of hepatocyte necrosis (←). On the 4^(th) day, the livers showed mild water degeneration (□) in hepatocytes around the central veins. From the 7^(th) day, liver lesions began to improve, the hepatic cords arranged radially centering the central veins and the hepatic sinusoids were clear. On the 14^(th) day, the livers also showed continuous improvement; necrosis was significantly reduced comparing with the first day, and the hepatic cords were more regular than the 7^(th) day.

In conclusion, in the PBS control group, the livers showed progressive damage from the first day to the 7^(th) day, and showed a tendency of improvement until the 14^(th) day, while in the administration group, the livers showed a significant improvement from the 7^(th) day, although the livers had a tendency of damage from the first day to the 4^(th) day. This indicates that plasminogen can promote the repair of liver damage caused by X-ray radiation.

Example 8: Effect of Plasminogen on Body Weight of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days, and the body weights were measured on the 0^(th) and 7^(th) days. The result showed that the mice in the solvent PBS control group were significantly reduced in body weight and had a statistically difference, while the mice in the plasminogen administration group had reduced body weight but the reduction was not significant (FIG. 8). This indicates that plasminogen can significantly reduce the effect of chemotherapeutic drug cisplatin on animal body weight.

Example 9: Protective Effect of Plasminogen on Kidneys of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days. The mice were killed on the 8th day and the kidneys were fixed in 10% neutral formalin for 24 to 48 hours. The fixed kidney tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The HE result showed that tubular epithelial cell necrosis (

) and inflammatory cell infiltration (←) were observed in the solvent PBS control group (FIG. 9A), while no significant necrosis was observed in the plasminogen administration group (FIG. 9B) and there was only a small amount of inflammatory cell infiltration. This indicates that plasminogen can reduce the kidney damage caused by chemotherapeutic drug cisplatin.

Example 10: Plasminogen Promoting Degradation of Fibrin in Kidneys of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days. The mice were killed on the 8th day and the kidneys were fixed in 10% neutral formalin for 24 to 48 hours. The fixed kidney tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to citric acid repair for 30 minutes, cooled at room temperature for 10 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse fibrin antibody (Abcam) and were washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abcam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

Fibrinogen is a precursor of fibrin, and in the presence of tissue damage, as a stress reaction to the body damage, fibrinogen is hydrolyzed into fibrin and deposited at the site of damage^([22-24]). Therefore, damage to local fibrin level can be used as a sign of the degree of damage.

The result showed that the positive staining of fibrin of the solvent PBS control group (FIG. 10A) was significantly deeper than that of the plasminogen administration group (FIG. 10B). This indicates that plasminogen can significantly reduce the fibrin deposition caused by chemotherapeutic drug cisplatin, and it is helpful for the repair of renal damage caused by the chemotherapeutic drug cisplatin.

Example 11: Plasminogen Promoting the Expression of Apoptosis Inhibitory Protein Bcl-2 in Kidneys of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days. The mice were killed on the 8th day and the kidneys were fixed in 10% neutral formalin for 24 to 48 hours. The fixed kidney tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to citric acid repair for 30 minutes, cooled at room temperature for 10 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse Bcl-2 antibody (Abcam) and were washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abcam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

Bcl-2 is an apoptosis inhibitory protein that has downregulation under the action of apoptosis induction[25, 26] The result showed that the positive staining of Bcl-2 in kidney tissues of the solvent PBS control group (FIG. 11A) was significantly lower than that of the plasminogen administration group (FIG. 11B). This indicates that plasminogen can significantly increase the expression of apoptosis inhibitory protein Bcl-2 in kidney tissues caused by chemotherapeutic drug cisplatin, which helps to inhibit the apoptosis of renal tissue cells.

Example 12: Protective Effect of Plasminogen on Livers of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. On the first day, intraperitoneal injection of cisplatin was started and plasminogen or solvent PBS was given. The administration period was 7 days. The mice were killed on the 8th day and the livers were fixed in 10% neutral formalin for 24 to 48 hours. The fixed liver tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The HE result showed that in the solvent PBS control group (FIG. 12A), the central vein of the liver was expanded, the endothelial cells were necrotic, and the surrounding had mild inflammatory cell infiltration. Also, large inflammatory lesions were visible (←), spotty necrosis of liver cells, nuclear fragmentation, cytoplasm light staining and hepatic disorders were observed. In the plasminogen administration group (FIG. 12B), the hepatic cords of the liver were scattered around the central vein with clear hepatic sinusoids and red staining of the cytoplasm, and necrosis of hepatocytes and infiltration of inflammatory cells were significantly reduced. This indicates that plasminogen can significantly reduce the liver damage caused by chemotherapeutic drug cisplatin.

Example 13: Plasminogen Promoting Degradation of Fibrin in Livers of Cisplatin-Chemotherapy-Injured Model Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days. The mice were killed on the 8th day and the livers were fixed in 10% neutral formalin for 24 to 48 hours. The fixed liver tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and washed once. They were subjected to citric acid repair for 30 minutes, cooled at room temperature for 10 minutes and then were gently rinsed by water. They were incubated with 3% hydrogen peroxide for 15 minutes and then tissues were circled with a PAP pen. They were blocked with 10% normal goal serum (Vector laboratories, Inc., USA) for 1 hour, and then the goat serum was discarded. They were incubated at 4° C. overnight with rabbit anti-mouse fibrin antibody (Abeam) and were, washed twice by TBS, 5 minutes each time. They were incubated at secondary anti-room temperature for 1 hour with goat anti-rabbit IgG (HRP) antibody (Abcam) and were washed twice by TBS, 5 minutes each time. The color was developed by DAB kit (Vector laboratories, Inc., USA) and they were washed three times with water. Then they were stained with hematoxylin for 30 seconds and rinsed with water for 5 minutes. They were dehydrated in gradient, transparentized and sealed. The slices were observed under the microscope at 200 times.

Fibrinogen is a precursor of fibrin, and in the presence of tissue damage, as a stress reaction to the body damage, fibrinogen is hydrolyzed into fibrin and deposited at the site of damage[22-24]. Therefore, damage to local fibrin level can be used as a sign of the degree of damage.

The result showed that the positive staining of fibrin in the liver tissues of the solvent PBS control group (FIG. 13A) was significantly deeper than that of the plasminogen administration group (FIG. 13B). This indicates that plasminogen can significantly reduce the fibrin deposition, which promotes the repair of liver damage caused by the chemotherapeutic drug cisplatin.

Example 14: Plasminogen Reducing Toxicity of Cisplatin Treatment on Reproductive Organs of Mice

In the present experiment, 10 healthy male C57 mice aged 8 to 9 weeks were randomly divided into two groups and given to the solvent PBS control group and the plasminogen administration group, 5 mice for each group. After grouping, a chemotherapy damage model was established and cisplatin was given via intraperitoneal injection for a single time at 10 mg/Kg body weight. After the model was established, plasminogen was administered to the plasminogen administration group via tail vein injection at 1 mg/0.1 mL/body/day, and the solvent PBS control group was given the same volume of PBS. The day when the experiment began was the 0^(th) day, and the body weights were weighed and grouped. Intraperitoneal injection of cisplatin was started on the first day, and plasminogen or solvent PBS was given within 3 hours after the model was established. The administration period was 7 days. The mice were killed on the 8th day and the testes and epididymides were fixed in 10% neutral formalin for 24 to 48 hours. The fixed testis and epididymis tissues were dehydrated by gradient alcohol, transparentized in xylene, and then paraffin-embedded. The thickness of tissue slices was 5 μm. The slices were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated by gradient alcohol and sealed. The slices were observed under the microscope at 200 times.

The result showed that in the solvent PBS control group, the number of spermatozoa in deferent duct of epididymis (FIG. 14A) decreased, accompanied by multiple epididymal tissue necrosis, and the number of spermatozoa was significantly reduced as compared with the plasminogen administration group, and multiple interstitial necrosis of the testis (FIG. 14C) was observed. In contrast with the solvent PBS control group, the number of spermatozoa in deferent duct of epididymis in the plasminogen administration group (FIG. 14B) was also significantly greater than that of the solvent PBS control group, and testis interstitial (FIG. 14D) necrotic lesions (single necrotic lesions) were also significantly reduced. This indicates that plasminogen can reduce the toxicity to the reproductive organs caused by chemotherapeutic drug cisplatin.

REFERENCES

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DRAWINGS

FIG. 1:

Weight (g)

0th day 5th day 12th day 21st day

FIG. 3:

Average optical density

FIG. 7:

1st day 4th day 7th day 14th day

FIG. 8:

Weight (g)

0th day 7th day 0th day 7th day 

1. A method for treating and/or eliminating radiation damage and chemical damage and related diseases in a subject, comprising administering to the subject an effective amount of plasminogen.
 2. A method for treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject, comprising administering to the subject an effective amount of plasminogen.
 3. The method according to claim 1, wherein the damage comprises damage to bone marrow hematopoietic system, skin, mucous membrane, immune system and reproductive system.
 4. The method according to claim 1, wherein the damage comprises damage to liver, spleen, kidney, lung, gastrointestinal tract, thymus, bone marrow, testis and epididymis.
 5. The method according to claim 1, wherein the damage comprises decrease in general healthy conditions, systemic side effects and local side effects.
 6. The method according to claim 1, wherein the damage-related diseases comprise mucosal ulcers, decreased immune function, myelosuppression, digestive dysfunction, heart, liver, spleen, lung, kidney, ovarian, testicular toxicity dysfunctions, and neurotoxicity dysfunction.
 7. The method according to claim 1, wherein the plasminogen has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2, 6, 8, 10 or 12 and still has plasminogen activity.
 8. The method according to claim 1, wherein the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
 9. The method according to claim 1, wherein the plasminogen is administered in combination with one or more other drugs or therapies.
 10. (canceled)
 11. An article for treating and/or eliminating body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject, comprising a container containing an effective amount of plasminogen, and an instruction for administering the article to treat and/or eliminate body organ and tissue damage and related diseases caused by radiotherapy, chemotherapy or chemoradiotherapy in a subject.
 12. The article according to claim 11, further comprising a container containing one or more other drugs.
 13. The article according to claim 12, wherein the other drugs comprise anti-cancer drugs, anti-infective drugs, immunopotentiators, analgesics, nutrients and antidotes.
 14. The article according to claim 11, wherein the instruction further states that the plasminogen is administered before, simultaneously with, and/or after the administration of the other drugs.
 15. The method according to claim 1, wherein the damage comprises acute side effects, long-term side effects and cumulative side effects caused by radiotherapy, chemotherapy or chemoradiotherapy.
 16. The method according to claim 9, wherein the other drugs or therapies comprise anti-cancer drugs, anti-infective drugs, immunopotentiators, analgesics, nutrients and antidotes. 